Segmentation of the early Drosophila embryo is accomplished through a cascade of genes that are precisely expressed in specific patterns. The boundaries that define the borders of these patterns are established very early during development with the remarkable spatial accuracy of a single cell diameter, representing the earliest evidence of developmental precision and reliability during embryogenesis. The exact molecular or biophysical mechanisms underlying the formation of boundaries and their accuracy are unknown. In general, precise morphogen gradients serve as transcriptional inputs that position a boundary, and mechanisms such as cooperativity or compensation are thought to help sharpen and maintain the boundary. Here we propose to put these hypotheses to a quantitative test by employing a combination of genetic experiments, precise measurements and mathematical modeling of the Bicoid morphogen gradient and its target genes in early Drosophila embryos. We have developed a new method to quantify mRNA of multiple genes at the single molecule level in whole embryos, which provides an approach to determine absolute numbers of both mRNAs and proteins in the same embryo. We will use this method in combination with live imaging of embryos expressing fluorescently tagged Bicoid to address the following questions: 1. how is a precise and stable transcriptional input achieved? 2. How does cooperativity of input factors activate and sharpen a boundary? 3. What are the responses of the system to gene dosage changes and what are the mechanisms that allow the embryo to compensate the response to such input changes? PUBLIC HEALTH RELEVANCE: Embryonic development is an extraordinary reliable process. It is robust against genetic and environmental perturbations, and it is highly reproducible from one embryo to the next. On the other hand, the tiniest error, down at the molecular level, can have the severest consequences, such as developmental or birth defects, and errors in developmental signaling pathways can lead to the formation of cancer. Hence, it is crucial to understand the mechanisms that underlie developmental reliability. Do developmental systems possess mechanisms for error correction, or are developmental processes very precise and reproducible at each stage? The answers to these questions are of molecular nature, and demand a quantitative approach that provides us with precise measurements of the molecular processes that govern development.